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Exp Neurol. Author manuscript; available in PMC 2018 February 01.
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Published in final edited form as:

Exp Neurol. 2017 February ; 288: 153–166. doi:10.1016/j.expneurol.2016.11.015.

Neurotoxic mechanisms of paclitaxel are local to the distal axon

and independent of transport defects
Erica L. Gornstein1,2 and Thomas L. Schwarz1
1The F.M. Kirby Neurobiology Center, Children’s Hospital Boston, Boston, MA 02115, USA
2Biological and Biomedical Sciences Program, Harvard Medical School, Boston, MA 02115, USA
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Abstract
Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of paclitaxel
and other chemotherapeutic agents. Paclitaxel binds and stabilizes microtubules, but the cellular
mechanisms that underlie paclitaxel’s neurotoxic effects are not well understood. We therefore
used primary cultures of adult murine dorsal root ganglion neurons, the cell type affected in
patients, to examine leading hypotheses to explain paclitaxel neurotoxicity. We address the role of
microtubule hyperstabilization and its downstream effects. Paclitaxel administered at 10–50 nM
for 1–3 days induced retraction bulbs at the tips of axons and arrested axon growth without
triggering axon fragmentation or cell death. By correlating the toxic effects and microtubule
stabilizing activity of structurally different microtubule stabilizing compounds, we confirmed that
microtubule hyperstabilization, rather than an off-target effect, is the likely primary cause of
paclitaxel neurotoxicity. We examined potential downstream consequences of microtubule
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hyperstabilization and found that changes in levels of tubulin posttranslational modifications,

although present after paclitaxel exposure, are not implicated in the paclitaxel neurotoxicity we
observed in the cultures. Additionally, defects in axonal transport were not implicated as an early,
causative mechanism of paclitaxel’s toxic effects on dorsal root ganglion neurons. By using
microfluidic chambers to selectively treat different parts of the axon with paclitaxel, we found that
the distal axon was primarily vulnerable to paclitaxel, indicating that paclitaxel acts directly on the
distal axon to induce degenerative effects. Together, our findings point to local effects of
microtubule hyperstabilization on the distal-most portion of the axon as an early mediator of
paclitaxel neurotoxicity. Because sensory neurons have a unique and ongoing requirement for
distal growth in order to reinnervate the epidermis as it turns over, we propose that the ability of
paclitaxel to arrest their growth accounts for the selective vulnerability of sensory neurons to
paclitaxel neurotoxicity.
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Keywords
paclitaxel; Taxol; chemotherapy-induced peripheral neuropathy; neurotoxicity; microtubule;
sensory neuron

Corresponding Author: Thomas L. Schwarz, Thomas.schwarz@childrens.harvard.edu, F.M. Kirby Neurobiology Center, Center for
Life Sciences, Rm. 12-130, 3 Blackfan Circle, Boston, MA 02115.
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Introduction
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Paclitaxel is a widely used chemotherapeutic that frequently causes peripheral neuropathy.

The neuropathy, which can be dose-limiting, primarily affects sensory neurons and leads to a
distal axonopathy; patients with paclitaxel-induced peripheral neuropathy experience loss of
sensation and ongoing pain in the hands and feet and demonstrate degeneration of the nerve
fibers that innervate the skin (1). Paclitaxel binds to the interior of microtubules and
stabilizes them, interfering with the normal cycling of microtubule depolymerization and
repolymerization. In dividing cells such as cancer cells, this disrupts normal spindle
dynamics, interferes with mitosis, and ultimately leads to cell death (2). Neurons, which do
not divide, are nevertheless susceptible to paclitaxel, and the mechanisms underlying
paclitaxel neurotoxicity remain incompletely elucidated (reviewed in (3)). Developing
interventions for paclitaxel-induced peripheral neuropathy will require understanding the
mechanisms by which paclitaxel compromises peripheral axons.
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Axons are rich in microtubules, which provide structural support and serve as tracks for
axonal transport throughout the life of the neuron. While neurons have a larger population of
stable microtubules than non-neuronal cells, their microtubules are not static. Given
paclitaxel’s action in dividing cells, it has been presumed that increased microtubule
stabilization contributes to paclitaxel neurotoxicity. However, alternative binding targets
leading to effects on the ER and mitochondria have been proposed that may be relevant (4–
6). Determining whether microtubule stabilization is the primary cause of paclitaxel
neurotoxicity is a first step in defining the mechanistic pathways leading to neuronal
damage.

If microtubule stabilization is the primary cause of paclitaxel neurotoxicity, the cellular

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mechanisms that link microtubule hyperstabilization and axon degeneration are unknown.
One known consequence of increased microtubule stabilization is a change in the levels of
tubulin posttranslational modifications, namely increased acetylation, polyglutamylation and
detyrosination (7–9). Tubulin post-translational modifications can alter the binding of
microtubule-associated proteins and motors, and alterations in these modifications have been
linked to axon degeneration and regeneration. For example, tyrosinated tubulin is necessary
for the binding of microtubule plus-end interacting proteins that are required for transport
initiation at the distal axon (10, 11). Tubulin hyperglutamylation has been linked to Purkinje
cell degeneration and increases the activity of the microtubule severing protein, spastin (12,
13). Additionally, microtubule deacetylation is required for axon regeneration after injury
(14), and kinesin-1 based transport is affected by acetylation and detyrosination (15–19).
Thus it is plausible that the alterations in tubulin post-translational modifications observed in
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neurons upon paclitaxel treatment contribute to paclitaxel neurotoxicity, a hypothesis which

has not yet been tested.

A prevalent hypothesis for how microtubule stabilization could ultimately lead to axon
degeneration is through the disruption of axonal transport. Previous studies have observed
paclitaxel-induced defects in axonal transport. These studies, however, mostly employed cell
types other than the clinically affected mammalian sensory neuron (20–22), or used
paclitaxel concentrations higher than the peak plasma concentrations typically reached in

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patients (23, 24). The relevance of potential transport defects as an early, causative event in
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paclitaxel neurotoxicity is uncertain.

Using cultured adult mammalian dorsal root ganglion (DRG) neurons, we examined the role
of microtubule stabilization and its downstream consequences in paclitaxel neurotoxicity.
Our results suggest that increased microtubule stabilization, rather than an off-target effect,
is likely to be responsible for paclitaxel neurotoxicity. However, our results do not point to
an early causative role of changes in tubulin post-translational modifications or axonal
transport defects in paclitaxel’s toxic effect on axons, but indicate instead a direct
vulnerability to paclitaxel of the distal-most portion of the axon. Inhibiting growth at the
distal axon may account for the sensory neuropathy.

Results
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Modeling paclitaxel neurotoxicity in cultured adult DRG neurons

In order to interrogate mechanisms of paclitaxel neurotoxicity, we established a model of
paclitaxel-induced degeneration in cultured adult mammalian DRG neurons, the neuron type
relevant to the clinical problem of paclitaxel-induced peripheral neuropathy. The use of
cultures necessarily restricted the study to cell autonomous aspects of the neuropathology
although other cell types are also likely to contribute (25, 26). For probing mechanisms of
paclitaxel neurotoxicity, we thought it important to use paclitaxel concentrations that
resulted in a slow time course of degeneration in order to untangle whether potential
mechanisms were a likely primary cause rather than indirect consequence of degeneration.
To this end, we used dissociated cultures of DRGs from 8–10 week old mice and exposed
them to nanomolar concentrations of paclitaxel that led to degenerative effects over the
course of days. Treatment of these sensory neuron cultures with 10–50 nM paclitaxel led
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within three days to a dose-dependent increase in bulbous swellings mostly at the axon tips,
an appearance consistent with retraction bulbs (Fig 1A–B). Axon area relative to untreated
cultures decreased with a similar time course (Fig 1C). After one day in 10 nM paclitaxel,
there were scattered bulbous axon swellings, which were more frequent upon exposure to 25
or 50nM paclitaxel. At each concentration, their frequency was further increased relative to
control after three days of paclitaxel exposure (Fig 1B). After three days in 25 nM or 50 nM
paclitaxel, axon area per field was also decreased, axon width was increased, and axons
appeared abnormally wavy. Nonetheless, the number of cell bodies per imaging field did not
change compared to control, indicating that in these treatment conditions, paclitaxel was
toxic to axons but did not cause cell death (Fig 1D). Because low nanomolar concentrations
of paclitaxel led to a slow time course of quantifiable degenerative effects, we used this
concentration range to probe paclitaxel neurotoxicity mechanisms. Peak plasma
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concentrations after common dosing regimens in patients are in the range of 228 nM-4.3 uM
(27), but the concentrations experienced by the neurons are not known. The range of 10–50
nM therefore seemed appropriate for dissociated DRG neurons in culture that likely have
better access and less transient exposure to the drug than neurons in vivo.

We characterized further the retraction bulb-like swellings that formed after paclitaxel
exposure. Unlike the growth cones of control axons, the retraction bulbs did not have actin
extending beyond the microtubules (Fig 1E). Microtubule polymerization was disordered in

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the retraction bulbs as evidenced by swirling EB3-GFP comets (Movie S1, Movie S2).
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Additionally, while the level of acetylated tubulin overall increased after paclitaxel
treatment, indicative of stable microtubules, tubulin in retraction bulbs was predominantly
deacetylated (Fig 1F). Furthermore, retraction bulbs had accumulations of mitochondria (Fig
1G). These observations are consistent with descriptions of CNS retraction bulbs, which are
characterized by disorganized microtubules (28).

We wondered whether neuronal types differed in their response to paclitaxel in vitro and
therefore whether neuronal type may be an important consideration when selecting a model
for mechanistic studies of paclitaxel neurotoxicity. We found that different neuron types had
different sensitivities and morphological responses to paclitaxel. In particular, cultured
hippocampal neurons were more sensitive than DRG neurons to paclitaxel when both were
exposed to 50 nM paclitaxel for three days. Microtubules were fragmented in hippocampal
neurons and their axons were no longer intact (Fig 1H). This fragmentation was not
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observed in DRG cultures, even after three days in 1 μM paclitaxel (data not shown). Prior to
fragmentation, hippocampal neurons did have some axonal swellings that might be akin to
the retraction bulbs of DRG neurons. After one day of 50 nM paclitaxel treatment, bulbous
structures were observed in hippocampal neurons near the soma at the tips of very short
neurites (data not shown); this response to paclitaxel has been previously reported at higher
concentrations in younger embryonic cortical neuron cultures (29). Previous work on
younger hippocampal cultures has shown that microtubule stabilization causes neurons to
form multiple axons when far lower doses of paclitaxel (3 nM) are applied (30). Adult DRG
axons are thicker, more robust, and more tubulin-rich than embryonic hippocampal neurons
and thus the different responses of the neurons to 50 nM paclitaxel may reflect either age-
dependent (31) or cell-type specific differences. The axonal effects seen in adult DRG
neurons after paclitaxel treatment better resemble the “dying back” axon retraction
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characteristic of paclitaxel-induced peripheral neurotoxicity (1) and thus may be preferable

to hippocampal neurons for mechanistic studies.

The neurotoxicity of epothilone B and paclitaxel correlates with their microtubule

stabilization
As a first step in defining mechanistic pathways that lead to paclitaxel neurotoxicity, we
tested the hypothesis that increased microtubule stability, rather than off-target effects, is the
primary cause of paclitaxel neurotoxicity. Although a change in microtubule dynamics after
paclitaxel exposure has been a dominant hypothesis to explain paclitaxel neurotoxicity,
alternative paclitaxel binding targets have been described: neuronal calcium sensor-1 and the
apoptosis regulator Bcl-2 (4–6). To test this hypothesis, we examined epothilone B, a
microtubule stabilizing compound that binds to a site on tubulin that overlaps the taxane site
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but is structurally different from paclitaxel; epothilone B would therefore be unlikely to have
the same off-target effects (32). If microtubule overstabilization is responsible for paclitaxel
neurotoxicity, it is expected that the prodegenerative capacity of paclitaxel and epothilone B
will correlate with the degree of microtubule stabilization. We examined microtubule
stability in cultured adult DRG neurons biochemically by assessing relative levels of soluble
βIII tubulin after treatment with 2 nM paclitaxel or epothilone B, replenishing the
compounds every 8 hours to maintain steady concentrations. 2 nM epothilone B strongly

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increased microtubule stability, as evidenced by a marked decrease in soluble βIII tubulin

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comparable to what is seen with 25 nM paclitaxel, but 2 nM paclitaxel was much less
effective (Fig 2A). This difference in potency at 2 nM allowed us to compare the
prodegenerative effects of the compounds. 2 nM epothilone B, which substantially stabilized
microtubules already at 24 hours, also significantly induced the incidence of retraction
bulbs, though 2 nM paclitaxel did not. After 72 hours, 2 nM epothilone B was also
significantly more potent in causing retraction bulbs (Fig 2B–C). In addition, 2 nM
epothilone B decreased axon area compared to control, while paclitaxel did not (Fig 2D–E).
While the axon area decreased after epothilone B treatment, the number of cell bodies per
field did not change, suggesting that, as with 25 nM paclitaxel, epothilone causes axonal
toxicity rather than cell death (Fig 2F). Overall, the morphological effects of epothilone B
and paclitaxel on DRG neurons were very similar, but epothilone B was roughly an order of
magnitude more potent than paclitaxel both in microtubule stabilizing activity and
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degenerative effects; neurons treated with 2 nM epothilone B resembled neurons treated with
25 nM paclitaxel. This correlation of microtubule stabilization and prodegenerative effects
for structurally dissimilar compounds supports the hypothesis that increased microtubule
stability, rather than an off-target effect, is responsible for paclitaxel neurotoxicity.

Changes in levels of tubulin modification do not correlate with retraction bulb formation
Why might increased microtubule stability be toxic to neurons? Stable microtubules have
increased levels of tubulin acetylation, polyglutamylation and detyrosination. These tubulin
post-translational modifications have been implicated in regulating axonal transport (15–19),
binding of microtubule-related proteins (11, 12), and neuron health (13, 14). We therefore
tested the hypothesis that changes in tubulin post-translational modifications contribute to
paclitaxel neurotoxicity as reflected in the formation of retraction bulbs; to this end, we
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manipulated the level or activity of the enzymes that control the cycling of tubulin
modifications.

Treatment of cultured DRG neurons with 25 nM paclitaxel led to an increase in

glutamylated tubulin as determined by immunocytochemistry and Western blot (Fig 3A–C).
By expressing shRNA against TTLL1, the catalytic subunit of the neuronal polyglutamylase,
control levels of glutamylated tubulin could be maintained even after paclitaxel treatment
(Fig 3A–C). Despite this rescue of glutamylation levels, the induction of retraction bulbs
after paclitaxel treatment was not ameliorated (Fig 3D–E). These data suggest that increased
tubulin glutamylation is not required for paclitaxel neurotoxicity.

25 nM paclitaxel also increased levels of acetylated tubulin in DRG axons (Fig 4A–B). To
determine whether this increase contributed to paclitaxel-induced toxicity, we asked whether
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increasing the acetylation of tubulin mimics paclitaxel induction of retraction bulbs. Neurons
were treated with the HDAC6 inhibitor tubacin to prevent deacetylation and thereby increase
tubulin acetylation to similar levels as caused by paclitaxel (Fig 4A–B). The paclitaxel and
tubacin increases in tubulin acetylation were also apparent in axons permeablized with triton
prior to fixation to extract soluble tubulin, indicating that the acetylated tubulin was
incorporated into microtubules (data not shown). Unlike paclitaxel, tubacin did not cause
retraction bulbs after either one or three days exposure (Fig 4C–D). That increased levels of

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acetylated tubulin were not sufficient to mimic paclitaxel’s effects on DRG axons suggests
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that the increase of acetylated tubulin is not a major contributor to paclitaxel neurotoxicity.

Stable microtubules have high ratios of detyrosinated to tyrosinated tubulin and DRG axons
treated with 50 nM paclitaxel have correspondingly decreased tyrosinated tubulin levels (Fig
5A–B). To ask if this decrease contributes to paclitaxel neurotoxicity, the tyrosinating
enzyme, tubulin tyrosine ligase (TTL) was overexpressed which caused control axonal levels
of tyrosinated tubulin to be maintained after paclitaxel exposure (Fig 5A–B). The tyrosinated
tubulin was resistant to triton permeabilization and therefore incorporated into microtubules
(data not shown). Despite preventing the paclitaxel-induced decrease in tyrosinated tubulin,
TTL expression did not prevent the formation of retraction bulbs (Fig 5C). Thus changes in
tubulin tyrosination are not required for paclitaxel neurotoxicity. Together, our
manipulations of tubulin post-translational modifications indicate that although tubulin
modifications are altered by paclitaxel treatment, they are unlikely to be responsible for the
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early paclitaxel-induced signs of neurodegeneration.

Effects of paclitaxel exposure on axonal transport of mitochondria and late endosomes/

lysosomes in cultured adult DRG neurons
A prevalent hypothesis to link microtubule stabilization and axon degeneration invokes
defects in axonal transport (33–35). We therefore asked whether axonal transport defects
arise in adult sensory neurons prior to overt fragmentation of axons and might thus be a
cause rather than consequence of neurotoxicity. To that end, 25 nM paclitaxel was applied to
adult DRG neurons, a condition in which axons remained intact but nonetheless developed
scattered retraction bulbs after one day and, after three days, had broader axons with more
retraction bulbs and more sparsely covered the field.
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Mitochondria are of particular interest to paclitaxel-induced neuropathy because swollen and

functionally deficient mitochondria have been observed in the peripheral nerves of animal
models (36, 37), and mitochondrial transport defects are implicated in several
neurodegenerative disorders (38). Mitochondria were therefore labeled with Mito-dsRed via
lentiviral transduction and their movements in a mid-axonal segment were imaged one and
three days after 25 nM paclitaxel treatment (Fig 6A–B). The percent time that mitochondria
spent in retrograde motion was slightly increased after one day of paclitaxel (Fig 6C),
however after three days of paclitaxel treatment there was no difference compared to vehicle
control in either anterograde or retrograde movement (Fig 6E). We did observe some
changes in mitochondrial velocities; retrograde speed was increased at both one and three
days of paclitaxel exposure, while anterograde speed was decreased, though only after one
day of paclitaxel treatment (Fig 6D, F). Peak plasma concentrations of paclitaxel in typical
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dosing regimens are in the range of 230 nM-4.3 μM (27). We therefore tested 250 nM and
2.5 μM paclitaxel for effects on mitochondrial transport after 6–9 hr of paclitaxel exposure,
after microtubules would be stabilized but prior to visible signs of neurotoxicity. Although
small changes in the parameters of movement were noted, they did not follow a clear dose-
response relationship (Fig 6G–J). For example, anterograde mitochondrial motility
decreased slightly and retrograde speed increased slightly with 250 nM paclitaxel, but these
differences were not observed with exposure to 2.5 μM paclitaxel (Fig 6G–J). Thus,

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although we looked over a broad range of concentrations and time points, paclitaxel’s effects
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on mitochondrial transport were modest and did not correlate well with the observed
toxicity; these data point away from a primary defect of mitochondrial transport as a cause
of paclitaxel neurotoxicity.

We also examined the effect of paclitaxel on transport of late endosomes and lysosomes by
expressing LAMP1-RFP. Because of the large number of fast-moving RFP-positive particles
in the axon, we analyzed organelle flux rather than the tracks of individual organelles. While
there was no difference in either anterograde or retrograde flux after one day of 25 nM
paclitaxel treatment, retrograde flux of lysosomes along the axon slightly decreased after
three days of paclitaxel treatment (Fig 7A–D). The observed changes in late endosomes and
lysosomes thus do not parallel the modest changes seen in mitochondrial transport; whereas
retrograde mitochondrial transport increased after one day of paclitaxel, retrograde flux
decreased for late endosomes/lysosomes after longer paclitaxel exposure.
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Exposure of the distal, but not the mid-axon, to paclitaxel disrupts axon outgrowth
Because paclitaxel treatment decreased axon area of cultured adult DRG neurons, we
examined the effects of paclitaxel on axon outgrowth by repeat imaging of GFP-expressing
neurons that had been cultured for six days and then exposed to 50 nM paclitaxel. Grooves
were scored into the culture substrate with a pin rake and dissociated neurons were plated as
a spot so that axons would extend in an orderly fashion between the grooves. This allowed
us to evaluate the growth of multiple axons in a single imaging field. At 2.5, 14 or 38 hours
after paclitaxel addition, a given field was imaged at 20 minute intervals for 100–120
minutes to evaluate the position of identified tips of axons. After just 2.5 hours of 50 nM
paclitaxel treatment, axon extension was reduced to 9.2% of vehicle treated control (Fig 8A,
D). This nearly complete arrest of axon outgrowth was also apparent 14 and 38 hours after
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paclitaxel addition (Fig 8B–C); many individual axons had no outgrowth or retracted their
axons during the 100–120 minutes of imaging. These results are consistent with the previous
finding that a higher concentration of paclitaxel, 700 nM, arrests outgrowth of one day old
chick sensory neurons in culture (39).

We used this assay to examine further the relevance of axonal transport defects to paclitaxel-
induced peripheral neuropathy and employed microfluidic devices to isolate paclitaxel
exposure to either mid-axons or distal axons. Dissociated DRG neurons were plated into the
somal compartment and axons grew through microgrooves into a 500 μm mid-axon
compartment and then through a second set of microgrooves into a 1500 μm distal
compartment (Fig 9A). Though the mid-axon compartment was smaller than the distal, the
axons were much less straight in the mid-axon channel, and therefore it is likely that
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comparable lengths of axon were exposed to paclitaxel when it was added to either
compartment. Trypan blue was used in pilot studies to confirm that fluidic isolation was
maintained over the course of the experiment. Mid or distal axons of adult DRG neurons
were selectively treated with 50 nM paclitaxel for two days after which multiple fields in the
distal compartment were imaged every 20 minutes for 120 minutes, as in Fig 8. The addition
of paclitaxel to the distal axon compartment decreased growth of distal axons; many axons
did not grow or retracted during the imaging period. In contrast, when the mid-axons were

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exposed to paclitaxel, the growth of the distal axons was not significantly different from
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control (Fig 9B). This result indicates that the distal axon in particular is vulnerable to
paclitaxel, and suggests that local effects of paclitaxel in the distal axon play a role in
paclitaxel neurotoxicity. Because growth cone dynamics depend on selective stabilization
and retraction of pioneer microtubules (40), the indiscriminate stabilization of microtubules
in the growth cone may be sufficient to explain the arrest of growth we observe.

Discussion
By using cultured DRG neurons to investigate paclitaxel-effects on axons, we could control
the level of paclitaxel exposure, limit it to restricted regions of the axon, monitor in detail
axonal transport, and manipulate biochemical pathways previously implicated in the toxicity
of chemotherapeutics. Although a murine model may imperfectly reflect events in human
neurons, we approximated the most clinically relevant population of cells by using DRG
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neurons from adult mice. We found that equivalent doses of paclitaxel had quite different
effects on E18 hippocampal neurons than on adult DRGs. Previous studies in cultures and in
vivo have gained significant insight into the mechanism of paclitaxel with systems as diverse
as Drosophila, Aplysia, and zebrafish, and with rodent neuronal populations from embryonic
DRG and hippocampi (3, 22, 25, 41, 42). By selecting cultured neurons for their advantages
in imaging and biochemical manipulations, we have necessarily restricted ourselves to cell
autonomous mechanisms and forgone the ability to examine the effects of paclitaxel on other
cell types that may contribute to neuropathy, including inflammatory responses and effects
on Schwann cells or epithelial targets (25, 26).

One difficulty in probing neurotoxic mechanisms has been distinguishing early, causative
effects from later consequences of neurodegeneration. We therefore used concentrations of
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paclitaxel that led to neurotoxic effects on DRG neurons, but did not cause rapid axon
fragmentation or cell death. Patients do not experience dramatic destruction of all sensory
axons, as can occur in response to very high concentrations of paclitaxel in other cell types,
but rather lose a portion of the sensory endings and usually after repeated paclitaxel
exposures (1). The changes in axons that we observed may therefore be similar to early
events that occur in patients. Although the concentrations used in this study are below
reported maximum plasma doses in patients (27), axons within the complex environment of
peripheral nerves are unlikely to experience the peak plasma concentration and those
concentrations are not sustained.

In DRG neurons exposed to paclitaxel, axon outgrowth stopped and bulbous endings known
as retraction bulbs formed. These endings contained dynamic, disorganized and deacetylated
microtubules and accumulations of mitochondria and presumably other organelles. We
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therefore used axon density and the frequency of retraction bulbs as a proxy for the dying
back neurodegenerative effect of paclitaxel in vivo.

Microtubule hyperstabilization is the best studied cellular action of paclitaxel and is often,
but not always (4, 35), presumed to underlie its neurotoxicity. We therefore compared the
efficacies of epothilone B and paclitaxel, two structurally unrelated stabilizers of
microtubules that likely would have different off-target effects. The degree of microtubule

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stabilization induced by these compounds correlated well with their prodegenerative

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capacity in cultured DRG neurons. Consistent with our observation of epothilone B as a

more potent microtubule stabilizer than paclitaxel, epothilone is generally more toxic than
paclitaxel to cancer cell lines (32). Moreover, treatment with ixabepilone, an analog of
epothilone B, also leads to a sensory peripheral neuropathy in patients (43). Our results
indicate that excess microtubule stabilization is likely to be the primary effect of paclitaxel
that initiates the downstream neurotoxic events. The WldS mouse and calpain inhibition
offer some protection against paclitaxel-induced degeneration (44, 45), and NMNAT
overexpression protects against the degeneration in a fly model (41); these interventions
likely block the downstream degeneration pathway triggered by microtubule stabilization.

What are the cellular mechanisms by which increased microtubule stabilization is

neurotoxic? We explored two current hypotheses: that changes in tubulin post-translational
modifications (46) or disruption of axonal transport are responsible for the degeneration.
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Though we confirmed that paclitaxel changes tubulin modifications in DRG neurons, the
changes in acetylation, glutamylation or tyrosination appeared unrelated to retraction bulb
formation. Manipulations that either mimicked the hyperacetylation, or prevented the
increase in glutamylated or decrease in tyrosinated tubulin did not mimic or prevent the
formation of retraction bulbs. Although there remains the possibility that a combination of
several of these changes would have uncovered an effect that our individual manipulations
did not, there was no noticeable decrease in the retraction bulb phenotype when tubulin
glutamylation and tyrosination changes were prevented. These posttranslational
modifications appear unlikely to cause the neurotoxicity in this system.

The length of sensory axons may make them highly dependent on microtubule-based
transport of organelles, proteins and mRNA. The vital role of axonal transport is
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underscored by mutations in motor proteins and defects in transport that are associated with
neurodegenerative diseases (47). We therefore looked closely at the effects of paclitaxel on
mitochondrial transport but did not observe any consistent defect in mitochondrial transport;
though minor changes in individual parameters were sometimes observed, none correlated
with either dose or duration and thus did not appear related to growth arrest or retraction
bulb formation. In neurodegenerative diseases that take decades to develop such as
Alzheimer’s disease, Amyotrophic Lateral Sclerosis, or Huntington’s disease, transport
perturbations too subtle to be detected by our assays might over time lead to axonal
degeneration. In this study, however, paclitaxel arrested axonal growth within 2.5 hours and
produced retraction bulbs within one to three days, and undetectable changes in transport are
therefore an unlikely cause.
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Previous studies of paclitaxel have observed bulk axonal transport defects at high paclitaxel
concentrations (10 uM and 200 uM, higher than the maximum plasma concentration
reported in patients) (23, 24). Studies using lower paclitaxel concentrations have also
observed bulk transport decreases (21, 22, 48) and mitochondrial transport decreases (20,
49), however these studies were not carried out on adult mammalian DRG neurons.

Selectively exposing either mid or distal axons to paclitaxel provided an independent

approach to interrogate a causative role of potential transport defects along the axon to

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paclitaxel neurotoxicity without presupposing which trafficked cargo would be most critical.
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We reasoned that a defect due to axonal transport should, given sufficient time of exposure,
manifest itself whether the block was close to the axon tip or in the middle of the axon. We
found that even two days of exposure of the medial portion of axon to paclitaxel did not
impede the growth of axons although comparable exposure of the distal axon did. These
findings again point away from axonal transport defects as an early event in paclitaxel
neurotoxicity.

A previous study in cultured neurons found that paclitaxel directly acts on peripheral
sensory axons rather than the soma: when embryonic DRG neurons were grown in
microfluidic devices such that paclitaxel could be selectively added to the axons or cell
bodies, axon length was reduced specifically when the axons were treated with paclitaxel
(42). Our study has taken the next step by comparing regions of the axon and has shown that
the distal portion of the axon is selectively sensitive. In contrast to the lack of an effect when
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paclitaxel was applied for two days but prevented from contacting the distal portion, arrest
of growth followed within 2.5 hours of exposure when the entire axon was exposed. The
results of the present study parallel those of Silva et al. (50), who found that, specifically at
low dose, the microtubule destabilizing agent vincristine was toxic when added to the distal,
but not the mid-axon, suggesting that a selective vulnerability of the distal axon could be a
commonality for microtubule-targeting agents. Furthermore, study of a low-dose rodent
model of paclitaxel-induced peripheral neuropathy found that degeneration was limited to
the sensory fibers innervating the epidermis and supports the particular sensitivity of these
distal nerve endings to paclitaxel (51).

Recent studies of growth cone dynamics have determined that microtubules play a key role
in both axon elongation and growth cone guidance (52, 53). Filipodial extension is driven by
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actin treadmilling, but microtubule growth and retraction, particularly for pioneer
microtubules, are also required for growth cone motility and consequently undergo extensive
regulation (40). Thus it is likely that indiscriminate stabilization of microtubules in growth
cones per se is sufficient to prevent axon elongation and induce retraction bulbs without
further changes in tubulin modifications or microtubule-based transport. In injured spinal
cord neurons, however, low doses of paclitaxel have been shown to enhance axon regrowth
and prevent retraction bulbs from forming (28, 54). In the context of injury when a glial scar
is forming and microtubules are destabilized, low doses of paclitaxel may restore correct
microtubule dynamics and axon extension. Thus the consequences of paclitaxel may be very
context dependent.

The selective vulnerability of the distal axon to paclitaxel in cultured neurons may arise
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from changes to microtubule dynamics in the growth cone and raises the question of whether
this is a phenomenon of relevance to mature sensory neurons in vivo that are not in a
comparable state of regeneration. Nerve fiber endings that innervate the skin are not,
however, static. It is estimated that the epidermis completely turns over in 40–56 days, and
in response nerve endings likely periodically remodel by withdrawing and then repenetrating
the layers of keratinocytes of the epidermis (51, 55, 56). GAP-43, a marker of axon growth
and plasticity, is present in a subset of nerve fibers in the dermis and epidermis in skin of the
adult hand (57). Thus sensory neurons may be dependent on axon extension and local

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 Gornstein and Schwarz Page 11

microtubule dynamics in vivo as well as in culture. Furthermore, a recent GWAS study has
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found an association in patients between genes involved in axon outgrowth and the severity
of paclitaxel-induced peripheral neuropathy (58). Maintenance of appropriate contact of
nerve fibers with the skin is important for continued retrograde survival signals at the distal
axon that play a role in maintaining axonal health (59). Thus, paclitaxel’s negative effect on
the plasticity of the distal axon suggests a hypothesis in which the inability to remodel in
response to the changing microenvironment of the epidermis in vivo could eventually lead to
neurite retraction and degeneration and may be an initial insult in paclitaxel-induced
degeneration. Furthermore, secondary to a neurite remodeling defect and retraction,
disruption of cellular processes at the distal axon, such as autophagosome formation (60)
and endocytosis of survival factors (59) could contribute to the pathogenesis.

One of the outstanding questions for chemotherapy-induced peripheral neuropathy has been
the selective vulnerability of sensory neurons over motor neurons. One current hypothesis is
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that the cell bodies of sensory neurons lie outside the blood brain barrier and are therefore
exposed to higher levels of the chemotherapeutic than those of motor neurons within the
spinal cord. Because we find that the distal axon is the site of paclitaxel action in cultured
sensory neurons, this hypothesis appears unlikely. Instead, we speculate that the difference
in vulnerability arises from the ongoing remodeling at adult sensory endings, which
contrasts with the stability of neuromuscular junctions. The initiating event is likely to be the
stabilization of microtubules in the growth cones of those sensory endings that need to
advance and reinnervate the epidermis during the administration of the drug. Because
microtubule stabilization is the mode of action for both the therapeutic and neurotoxic
actions of paclitaxel and related compounds, strategies to alleviate the neurotoxicity may
need to focus on downstream pro-degenerative events.
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Materials and Methods

Neuron culture
Dorsal root ganglia (DRG) from 8–10 week old male C57Bl/6J mice (Jackson labs) were
dissected into cold HBSS (Life Technologies) containing 1% penicillin-streptomycin (Life
Technologies) and dissociated in 1 mg/mL Collagenase A + 2.4 U/mL dispase II (Roche) for
70 min at 37°C, followed by 5 min in 0.25% trypsin (Life Technologies) at 37°C. After
addition of trypsin inhibitor (Sigma) and washes, DRG were triturated using glass pasteur
pipettes of decreasing size in the presence of 125U DNAse I (Sigma) and centrifuged
through 10% BSA (Sigma). The cell pellet was washed and then resuspended in Neurobasal
A (Life Technologies) with 2% B27 (Life Technologies), 100 U/mL penicillin-streptomycin,
1 mM L-glutamine (Life Technologies), 100 ng/μl NGF (Life Technologies), and 2 ng/μl
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GDNF (Sigma). 12–18 × 103 neurons/well were plated on acid washed glass coverslips
(Bellco Glass) in a 24-well plate that had been coated overnight with 100 μg/ml poly-d-
lysine (Sigma) at room temperature and 10 μg/ml laminin (Life Technologies) for 2 hr at
37°C. Beginning on DIV 1, 10 μM AraC (Sigma) was added to media.

Hippocampal neurons were isolated from embryonic day 18 rats (Charles River) as
previously described (61), and plated at 8 × 104/well in a 24-well plate on acid washed
coverslips that had been coated overnight at room temperature with 20 μg/ml poly-L-

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 Gornstein and Schwarz Page 12

ornithine (Sigma) and 3.5 μg/ml laminin. Cells were cultured in Neurobasal with 2% B27,
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supplemented with L-glutamine and penicillin-streptomycin.

Spot-Groove Culture
To grow straight axons for live imaging of distal axon endings, an optical-quality 24-well
plate (Ibidi) was coated with poly-d-lysine and laminin as described above for DRG neuron
culture. After coating, grooves were etched into each well using a pin rake (Tyler Research).
DRG from 8–10 week old male mice were dissected and dissociated as described above and
spotted in the center of the grooves at 15 × 103 cells/well in 7 μl of media. After allowing
cells to attach for 1 hr in the incubator, media (Neurobasal A, 2% B27, 100 U/mL penicillin-
streptomycin, 1 mM L-glutamine, 100 ng/μl NGF, 2 ng/μl GDNF) was slowly added.
Beginning on DIV 1, 10 μM AraC was added to media. For live imaging of distal axons,
cultures were infected with GFP-ires-GFP lentivirus at DIV 0, and cultures were grown for 6
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DIV before treatment with paclitaxel.

Microfluidic devices
Acid washed coverslips (Warner Instruments 24×50 mm) were coated with 100 μg/ml poly-
d-lysine overnight at room temperature. After washes with ddH2O, TCND500 microfluidic
devices (Xona Microfluidics) were attached to coverslips, and laminin was added to the
channels at a concentration of 10 μg/ml for 3 hr at 37°C. DRG from 8–10 week old male
mice were dissected and dissociated as described above and plated at 8 × 104 cells/
microfluidic device in 5 μl of media. After allowing cells to attach for 55 min in the
incubator, media (Neurobasal A, 2% B27, 100 U/mL penicillin-streptomycin, 1 mM L-
glutamine, 50 ng/μl NGF, 1 ng/μl GDNF) was added slowly to the cell body wells. A portion
of the cells in each device washed away when media was added. Media with 100 ng/μl NGF
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and 2 ng/μl GDNF was added to the axonal wells. Starting on DIV 1, 10 μM AraC was
added to media. Media was changed every 2 days until DIV 6, when axons were treated with
paclitaxel. Paclitaxel-containing media was replenished after 1 day.

Constructs and Compounds

The following lentiviral constructs were used in this study: EB3-GFP (62), and TTLL1-
specific shRNA and control vector (Rogowski et al., 2010). TTL-ires-GFP, which was
generated by cloning the coding sequence of human TTL from cDNA (GenBank BC036819)
into pHAGE-CMV-ires-GFP (63). GFP-ires-GFP was used as a control plasmid. Mito-
dsRed-ires-GFP, which was generated by cloning Mito-dsRed (Clontech) into pHAGE-
CMV-ires-GFP. LAMP1-RFP-2A-GFP, which was generated by cloning LAMP1-RFP
(Addgene #1817) into pLenti-CMV-2A-GFP (gift from C. Woolf).
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Paclitaxel (Sigma), (−)-Epothilone B (Sigma) and Tubacin (Enzo Life Sciences) were kept
as stocks in DMSO and were diluted in culture medium immediately prior to addition to
cells. Control cultures always received an equivalent amount of DMSO.

Lentivirus preparation
Lentiviral particles were produced by cotransfection of lentiviral expression vectors with
packaging helper plasmids into HEK293T cells plated in 15 cm2 dishes. Media containing

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 Gornstein and Schwarz Page 13

viral particles was collected 48 and 72 hr after transfection. After filtration through a 0.45
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μm filter, lentiviral particles were concentrated by spinning at 25,900 RPM at 4°C for 90
min (SW 32 Ti rotor, Beckman Coulter). The resulting viral pellet was resuspended in PBS/
0.001% Pluronic F68 (Sigma), concentrating 800-fold from the packaging cell media, and
stored at −80°C until use. Lentivirus was added to 250 μL culture media 3–4 hr after plating,
and media was changed 16–18 hr later. Expression of fluorescent protein in the neuron cell
body was used to monitor lentivirus expression.

Immunoflourescence
DRG neurons were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with
0.1% triton-X in PBS for 10 min, and blocked in 15% goat serum/1% BSA/0.1% triton-X in
PBS. Primary and secondary antibodies were diluted in block and incubated for 1 hr each at
room temperature. The following primary antibodies were used: mouse anti-βIII tubulin
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(1:800, Sigma), rabbit anti-TUJ1 (1:1000; Covance), chicken anti-GFP (1:500; Aves Labs),
mouse anti-glutamylated tubulin GT335 (1:3000; AdipoGen), mouse anti-acetylated tubulin
6-11B-1 (1:1000; Sigma), rat anti-tyrosinated tubulin YL1/2 (1:1000; Abcam), rabbit-anti
Tom20 (1:500; Santa Cruz Biotechnology). The following secondary antibodies were used at
1:500: AlexaFluor-488 anti-mouse, anti-rabbit, or anti-chicken, AlexaFluor-568 anti-rabbit,
AlexaFluor-647 anti-mouse (Invitrogen), Cy3-conjugated anti-mouse, Cy5-conjugated anti-
rat (Jackson ImmunoResearch). Images of fixed cells were acquired on a Nikon Eclipse
E800, or when fluorescence was to be quantified, a Zeiss LSM 710 confocal microscope.

Soluble tubulin and actin extraction assay

Dissociated DRG were cultured directly on poly-d-lysine and laminin coated 24-well plates.
After a wash with warm PBS, soluble protein was extracted by adding 100 μl microtubule
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stabilizing buffer (80 mM PIPES pH 6.8, 1 mM MgCl2, 1 mM EGTA, 5% glycerol, 0.5%

triton-X) containing 1 mM PMSF and protease inhibitor cocktail (Calbiochem) at 1:500 for
2 min at 37°C. Samples were centrifuged at 13,000 g for 10 min at 4°C, the supernatants
were collected and Laemmli buffer was added to 1x. An equal volume of each sample was
run for Western blot analysis.

Western blotting
Protein lysates were separated by 8% SDS-PAGE and blotted with the following primary
antibodies: rabbit anti-TUJ1 (1:2500; Covance), mouse anti-β-actin AC-74 (1:5000; Sigma),
mouse anti-glutamylated tubulin GT335 (1:2000; AdipoGen). HRP-conjugated secondary
antibodies (1:5000; Jackson ImmunoResearch) and SuperSignal West Dura (Thermo
Scientific) were used for chemiluminescent detection.
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For assessing levels of glutamylated tubulin in DRG cultures, cells grown directly on poly-d-
lysine and laminin coated 24-well plates were lysed in 1X Laemmli sample buffer and an
equal volume of each sample was loaded onto the SDS-PAGE gel.

Live cell time lapse imaging acquisition and quantification

For live cell imaging of transport of mitochondria or late endosomes/lysosomes in DRG
neurons, coverslips were transferred to petri dishes containing Hibernate A (BrainBits), with

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 Gornstein and Schwarz Page 14

2% B27 and 2 mM Glutamax (Life Technologies). DMSO or paclitaxel was added to the
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imaging media at the same concentration as was present in the growth media. Time lapse
movies were acquired on a Zeiss LSM 710 confocal microscope using a 63×/N.A.0.90 water
IR-Achroplan objective. Cells were maintained at 37°C on a temperature-controlled stage
(PeCon, GmbH). For imaging of mitochondria and late endosomes/lysosomes, portions of
axon 100 μm long were selected and traced at least 500 μm away from the cell body using
the GFP signal. Mitochondria were imaged at 1 frame/1.58 sec for 5 min, and late
endosomes/lysosomes for 3 min. EB3-GFP was imaged at 1 frame/3.9 sec for 3 min. Live
imaging of mitochondria and late endosomes/lysosomes was performed on DIV 4 and 6, and
imaging of EB3-GFP was performed on DIV 4.

The mitochondrial movement parameters “percent time in motion per axon” and “average
speed per mitochondrion” were quantified in ImageJ. Kymographs were generated from
time lapse movies after running the Image Stabilizer plugin when necessary and
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mitochondria tracks were analyzed by manual tracing using a custom macro, Kymolyzer, as
previously described (64). The percentage of time mitochondria were in anterograde or
retrograde motion per axon was calculated by averaging the percent of frames in a trace for
which each mitochondrion moved (ranging from 0–100%) in a kymograph. An average
speed for each mitochondrion was calculated as the average of all instantaneous speeds in a
trace that were not zero. The overall average was then calculated using the data from each
mitochondrion. Movement slower than 0.05μm/sec was considered zero.

To determine lysosome flux, a vertical line was drawn near the center of the generated
kymograph, and the number of tracks crossing the line in the anterograde and retrograde
direction were counted.
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For live imaging of axon growth in spot-groove cultures, DIV 6 DRG neurons were treated
with paclitaxel or DMSO vehicle in phenol red-free complete growth media. Images of axon
endings expressing GFP were acquired using the 40× objective on a Nikon Eclipse Ti
microscope with an environmental chamber. Axon fields for imaging were selected prior to
paclitaxel treatment, and the same axon fields were imaged 2.5, 14 and 38 hr after paclitaxel
treatment for 100–120 min, with an image captured every 20 min. Axon tips analyzed for
growth were selected prior to visualizing the movies, and a line was drawn using ImageJ at
the point reached by the axon tip at t=0 for each imaging session. The distance of the axon
tip from this line was measured at each 20 min interval of the 100–120 min imaging period.

For live imaging of axon growth in microfluidic chambers, DIC images of axons in the distal
channel were acquired using a 40× objective on a Nikon Eclipse Ti microscope with an
environmental chamber. Multiple fields containing axonal endings were imaged repeatedly
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every 20 min for 120 min. Distal axon growth was analyzed as described above for spot-
groove cultures.

Image analysis
To quantify changes in levels of modified tubulin by immunofluorescence, maximum
intensity projections were created of images taken with a 63× objective using identical
imaging settings. To limit quantification of fluorescence to neuronal cells and to remove

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 Gornstein and Schwarz Page 15

background, using ImageJ, the Huang threshold was applied to the βIII tubulin staining, and
the mean intensity of acetylated, glutamylated, tyrosinated, or βIII tubulin within the
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thresholded βIII tubulin outline was calculated. The intensity from modified tubulin was
divided by the βIII tubulin intensity for each condition. The average value from 3–4 imaging
fields for each condition from each experiment was used for statistical analysis.

The number of retraction bulb-like swellings and the number of cell bodies per field was
counted manually from images taken with a 10× or 20× objective using the Cell Counter
plugin in ImageJ.

Neurite area per field was quantified with ImageJ from images taken with a 20× objective
using a macro based on Pani et al. (65) for assessing neurite area in dense cultures with both
dim and bright neurites. In summary, after background subtraction, contrast enhancement,
and application of a Gaussian blur filter, cell bodies were deleted manually from the image.
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Next, edges were enhanced using the FeatureJ Laplacian plugin. After contrast enhancement
and application of the Moments threshold, a mask was created, and the analyze particles
function (size: 50-infinity) was used to determine the area of the axon. For axon area and
retraction bulb quantification, 4–6 imaging fields per coverslip and 2 coverslips from each
condition were quantified per experiment, and the average value from each experiment was
used for statistical analysis.

Statistics
Data are expressed as mean ± SEM. Statistical analysis was performed using GraphPad
Prism 6. The unpaired Student’s t test or the Mann-Whitney U test was used assess the
statistical significance of differences between conditions. For multiple comparisons, data
were analyzed by two-way ANOVA with Bonferroni correction or Dunnett’s post test.
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p<0.05 was considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
This work was supported by National Institutes of Health R01GM069808 (T.L. Schwarz) and F31 NS089152 (E.L.
Gornstein), and the Mather’s Foundation (T.L. Schwarz).

The authors thank C. Janke, M. Verhage, C. Woolf, and M. Sahin for constructs; T. Omura, C. Su, and M. Costigan
for technical advice on DRG cultures; S. Vasquez, K. Apaydin, and L. Mkhitaryan for assistance with hippocampal
neuron cultures; and the CHB IDDRC Imaging Core (grant P30 HD18655).
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Fig 1. Effects of paclitaxel exposure on cultured adult DRG neurons

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A) In DRG cultures immunostained for βIII tubulin, swellings (blue arrowheads) are present
at axon tips after 25 nM paclitaxel treatment for 72 hr starting at 4 DIV, but not in control
cultures. Scale bar, 20 μm. Insets compare a normal growth cone in control cultures and
paclitaxel-induced retraction bulbs. Scale bars, 5 μm.
B–D) Quantification of DRG neurons treated with paclitaxel at the indicated concentrations
for 24 or 72 hr, beginning at 4 DIV and stained for βIII tubulin. In each case, values for
control cultures plated the same day were set as 1 and fold changes in response to paclitaxel
treatment were calculated. n=3 experiments. (B) Number of retraction bulbs per field.

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Relative to control, p=0.019 (10 nM), p=0.0078 (25 nM), p=0.033 (50 nM) at 24 hr, and
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p=0.038 (10 nM), p=0.012 (25 nM), p<0.0001 (50 nM) at 72 hr; Student’s t test. (C) Axon
area at 72 hr was calculated from masks created from original images. **p<0.01,
***p<0.001; Student’s t test. (D) Number of cell bodies per field. p>0.05, Student’s t test.
E) A control growth cone and a paclitaxel-induced retraction bulb stained for F-actin with
rhodamine-phalloidin and for βIII tubulin after three days of treatment. Scale bar, 5 μm.
F–G) Paclitaxel-treated DRG neurons immunostained for βIII tubulin and acetylated tubulin
(F) or the mitochondrial marker Tom20 (G) after three days of treatment. Retraction bulbs
had no detectable acetylated tubulin but a concentration of mitochondria. Scale bars, 10 μm.
H) Hippocampal neurons (7 DIV) and DRG neurons (4 DIV) were each treated with 50 nM
paclitaxel for 3 days and then stained for βIII tubulin. Paclitaxel induced greater
degeneration in hippocampal neurons Scale bar, 20 μm.
Ptx, paclitaxel.
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Fig 2. The neurotoxicity of epothilone B and paclitaxel correlates with their microtubule
stabilization
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Cultured DRG neurons were treated with 2 nM paclitaxel or epothilone B starting at 3 DIV
and treatment was refreshed every 8 hr for 24 or 72 hr.
A) After epothilone B treatment, lower levels of soluble tubulin remained than after
paclitaxel due to the greater potency of epothilone at stabilizing polymerized tubulin. Actin
serves as a loading control.
B) Quantification of retraction bulbs per field. The number of retraction bulbs in control
cultures was set as 1 and fold changes in response to paclitaxel and epothilone B treatment
were calculated. ***p<0.001, ****p<0.0001; Student’s t test.

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C) Representative fields immunostained for βIII tubulin with retraction bulbs (blue
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arrowheads) at 72 hr, as quantified in (B). Scale bar, 50 μm.

D–E) To assess axon area after 72 hr of treatment, cell bodies were removed from the
images and an axon mask was created. Axon area was then quantified and expressed as fold
change compared control. Scale bar, 100 μm. **p<0.01, Student’s t test.
F) The number of cell bodies per field was counted from images like those in (D) and
expressed as fold change compared to control. p>0.05, Student’s t test.
For (A–F), n=4 experiments. Ptx, paclitaxel.
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Fig 3. Maintaining control levels of glutamylated tubulin after paclitaxel treatment does not
rescue paclitaxel neurotoxicity
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A–B) DRG cultures were infected with TTLL1 shRNA lentivirus or control vector at 0 DIV,
treated for 24 hr with 25 nM paclitaxel at 6 DIV, and immunostained for βIII tubulin and
glutamylated tubulin. The fluorescence intensity of glutamylated tubulin signal from axons
was normalized to βIII tubulin signal, and fold changes relative to control were calculated.
Scale bar, 20 μm. p=0.026 (Vector vs. Vector 25 nM), p=0.037 (Vector 25 nM vs. TTLLsh
25 nM), ns p=0.92; Student’s t test.

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C) Glutamylated tubulin and βIII tubulin in Western blots of lysates of DRG cultures as in
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(A). TTLL1 shRNA reduced tubulin glutamylation in the presence of paclitaxel to control
levels.
D–E) DRG cultures as in (A) and immunostained for βIII tubulin show retraction bulbs after
paclitaxel treatment whether or not glutamylation was inhibited. Cell bodies are indicated by
asterisks (*). Number of retraction bulbs per field is expressed as a fold change relative to
control. Scale bar, 50 μm. **p<0.01, ns p=0.75; Student’s t test.
For (A–E), n=3 experiments. Ptx, paclitaxel.
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Fig 4. Increasing acetylated tubulin with tubacin does not mimic paclitaxel neurotoxicity
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DRG neurons at 3 DIV were treated with 20 μM tubacin or 25 nM paclitaxel for 24 or 72 hr

refreshed every 24 hr.
A–B) The immunofluorescence intensity of acetylated tubulin in axons was normalized to
the βIII tubulin immunofluorescence, and fold changes from control were calculated. Scale
bar, 20 μm. Relative to control, p=0.0049 (25 nM Ptx), p=0.036 (tubacin) at 24 hr, and
p=0.010 (25 nM Ptx), p=0.047 (tubacin) at 72 hr; Student’s t test.
C–D) 25 nM paclitaxel but not 20 μM tubacin treatment caused retraction bulbs to form as
visualized with anti-βIII tubulin (C) and quantified per field (D). Scale bar, 50 μm. Cell

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bodies are indicated by asterisks (*). Relative to control, p=0.038 (25 nM Ptx), p=0.46
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(tubacin) at 24 hr, and p=0.011 (25 nM Ptx), p=0.35 (tubacin) at 72 hr; Student’s t test.
For (A–D), n=3 experiments. Ptx, paclitaxel.
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Fig 5. Maintaining control levels of tyrosinated tubulin after paclitaxel treatment does not rescue
paclitaxel neurotoxicity
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TTL or GFP control lentivirus was added to DRG neurons at 0 DIV and treated with 50 nM
paclitaxel for 48 hr starting at 5 DIV prior to immunostaining for tyrosinated tubulin and
βIII tubulin at 7 DIV.
A–B) The fluorescence intensity of tyrosinated tubulin signal from axons was normalized to
βIII tubulin signal, and fold changes from control were calculated. Scale bar, 20 μm.
**p<0.01, ns p=0.18; Student’s t test.
C) Number of retraction bulbs per field was quantified and is expressed as a fold change
relative to control. ***p<0.001, *p=0.037, ns p=0.28; Student’s t test.
For (A–C) n= 3 experiments. Ptx, paclitaxel.
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Fig 6. Axonal transport of mitochondria persists after paclitaxel exposure

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A–B) Kymographs of mitochondrial movement in DRG axons infected with Mito-DsRed

lentivirus and treated with DMSO as a control (A) or 25 nM paclitaxel (B) for 1 day prior to
imaging at 4 DIV. Kymographs represent position (x axis) over time (y axis) such that
vertical lines indicate stationary mitochondria and diagonal lines indicate moving
mitochondria. The image above the kymograph is the first frame of the time lapse movie
used to generate the kymograph. Scale bars, 10 μm 100 s.
C) The percent of time that mitochondria moved in either the anterograde or retrograde
direction per axon after treatment with DMSO control or 25 nM paclitaxel for 1 day was

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quantified using kymographs like those in (A) and (B). n=23–24 axons from 4 independent
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experiments. *p=0.026, Student’s t test.

D) The average speed of each mitochondrion moving either anterograde or retrograde after
treatment with DMSO control or 25 nM paclitaxel for 1 day was quantified using
kymographs like those in (A) and (B). n=371–410 (anterograde) and n=222–322
(retrograde) mitochondria from 4 experiments. *p=0.029, **p<0.01; Mann-Whitney U test.
E) Quantification of the percent of time mitochondria were moving after treatment with
DMSO control or 25 nM paclitaxel for 3 days, replenished every 24 hr. n=19–21 axons from
4 experiments. p>0.05, Student’s t test.
F) Quantification of the average speed of each mitochondrion after treatment with DMSO
control or 25 nM paclitaxel for 3 days, replenished every 24 hr. n=191–214 (anterograde)
and 195–212 (retrograde) mitochondria from 4 experiments. ***p<0.001, Mann-Whitney U
test.
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G–J) Quantification of mitochondrial parameters as in (C–F), but in the presence of higher

paclitaxel concentrations for 6–9 hr. (G–H) n=20–31 axons from 5 experiments. *p=0.02,
Student’s t test. (I) n=241–309 (anterograde) and n=160–256 (retrograde) mitochondria
from 5 experiments. ***p<0.001, Mann-Whitney U test. (J) n=246–292 (anterograde) and
n=198–207 (retrograde) mitochondria from 5 experiments. *p=0.047, Mann-Whitney U test.
Ptx, paclitaxel.
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Fig 7. Effect of paclitaxel on lysosome transport

A–B) Kymographs of lysosome movement in DRG axons infected with LAMP1-RFP
lentivirus and treated with DMSO as a control (A) or 25 nM paclitaxel (B) for 3 days, and
imaged at 6 DIV. The image above the kymograph is the first frame of the time lapse movie
used to generate the kymograph. Scale bars, 10 μm 100 s.
C–D) Average anterograde and retrograde lysosome flux per axon after treatment with
DMSO control or 25 nM paclitaxel for 1 day (C) or 3 days (D). Flux was quantified from
kymographs like those in (A) and (B) by counting LAMP1-RFP tracks that crossed a line
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drawn in the center of the kymograph. n=17–19 axons from 3 experiments. **p<0.01,
Student’s t test.
Ptx, paclitaxel.
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Fig 8. Paclitaxel quickly and stably inhibits DRG axon growth

DRG neurons infected with GFP lentivirus at DIV 0 were grown 6 days in culture and then
treated with 50 nM paclitaxel. Images of the same fields of GFP+ distal axons were taken
every 20 min for 100–120 min at 2.5 hr, 14 hr, and 38 hr after paclitaxel or DMSO control
treatment.
A–C) The distance in microns that each axon ending moved (grew or retracted) relative to
t=0 was measured for each time interval. n=41–50 axons from 2 experiments.
D) Temporal color coding of the 100 min time lapse beginning 2.5 hr after paclitaxel
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addition. Arrowheads indicate the starting points of the indicated axon endings. While axon
growth occurred in the control treatment, little to no growth or retraction was seen after
paclitaxel treatment. Scale bar, 20 μm.
*p<0.05, ***p<0.001, ****p<0.0001; two-way ANOVA with Bonferroni correction. Ptx,
paclitaxel.
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Fig 9. Paclitaxel exposure of the distal axon, but not the mid-axon, disrupts axon outgrowth
A) Schematic of the use of microfluidic devices to examine the effects of exposing either the
distal portion of the axon or a mid-axonal segment to paclitaxel. The culture chamber
consists of 3 fluidically isolated compartments (cell body, mid-axon and distal axon)
connected by 2 sets of microgrooves. Starting at 6 DIV, either the mid-axon or distal axons
were exposed to 50 nM paclitaxel for two days after which distal axons were imaged every
20 min for 120 min.
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B) The distance in microns that each axon ending moved (grew or retracted) relative to t=0
was measured for each experimental condition. n= 252–308 axons from 4–5 experiments per
condition.
****p<0.0001, ns p>0.05; two-way ANOVA, Dunnett’s post test. Ptx, paclitaxel.
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